The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (kcat/KM) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered KM for manno-oligosaccharides by 30–45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, KM for ManC W283S was increased, the transglycosylation yield was reduced by 30–45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.